title: The three α1-adrenoceptor subtypes show different spatio-temporal mechanisms of internalization and ERK1/2 phosphorylation.
abstract: We analyzed the kinetic and spatial patterns characterizing activation of the MAP kinases ERK 1 and 2 (ERK1/2) by the three α1-adrenoceptor (α1-AR) subtypes in HEK293 cells and the contribution of two different pathways to ERK1/2 phosphorylation: protein kinase C (PKC)-dependent ERK1/2 activation and internalization-dependent ERK1/2 activation. The different pathways of phenylephrine induced ERK phosphorylation were determined by western blot, using the PKC inhibitor Ro 31-8425, the receptor internalization inhibitor concanavalin A and the siRNA targeting β-arrestin 2. Receptor internalization properties were studied using CypHer5 technology and VSV-G epitope-tagged receptors. Activation of α1A- and α1B-ARs by phenylephrine elicited rapid ERK1/2 phosphorylation that was directed to the nucleus and inhibited by Ro 31-8425. Concomitant with phenylephrine induced receptor internalization α1A-AR, but not α1B-AR, produced a maintained and PKC-independent ERK phosphorylation, which was restricted to the cytosol and inhibited by β-arrestin 2 knockdown or concanavalin A treatment. α1D-AR displayed constitutive ERK phosphorylation, which was reduced by incubation with prazosin or the selective α1D antagonist BMY7378. Following activation by phenylephrine, α1D-AR elicited rapid, transient ERK1/2 phosphorylation that was restricted to the cytosol and not inhibited by Ro 31-8425. Internalization of the α1D-AR subtype was not observed via CypHer5 technology. The three α1-AR subtypes present different spatio-temporal patterns of receptor internalization, and only α1A-AR stimulation translates to a late, sustained ERK1/2 phosphorylation that is restricted to the cytosol and dependent on β-arrestin 2 mediated internalization.
authors.affiliation: Departament de Farmacologia, Universitat de València, Spain.
authors.forename: M
authors.initials: M
authors.lastname: Perez-Aso
authors.forename: V
authors.initials: V
authors.lastname: Segura
authors.forename: F
authors.initials: F
authors.lastname: Montó
authors.forename: D
authors.initials: D
authors.lastname: Barettino
authors.forename: M A
authors.initials: MA
authors.lastname: Noguera
authors.forename: G
authors.initials: G
authors.lastname: Milligan
authors.forename: P
authors.initials: P
authors.lastname: D'Ocon
mesh_terms: D019390:Arrestins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D002478:Cells, Cultured; D003208:Concanavalin A / Q000494:pharmacology; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007124:Immunoenzyme Techniques; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D018340:Receptors, Adrenergic, alpha-1 / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins
chemical_list: C000605254:ARRB2 protein, human; D019390:Arrestins; D004791:Enzyme Inhibitors; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D018340:Receptors, Adrenergic, alpha-1; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; D003208:Concanavalin A; D011493:Protein Kinase C; C535150:MAPK1 protein, human; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3
keywords: AT(1A)R; Adrenaline α(1) receptors; BSA; Bmax; ConA; Constitutive activity; DMSO; EGFR; ERK1/2; G-protein coupled receptor; G-protein-coupled receptor kinase; GAPDH; GPCR; GRK; HEK; Internalization; KRH; Krebs Ringer Hepes; MAPK; PBST; PCR; PE; PKA; PKC; PTHR; PZ; angiotensin receptor type 1A; bovine serum albumin; concanavalin A; dimethyl sulfoxide; epidermal growth factor receptor; glyceraldehyde-3-phosphate dehydrogenase; human embryonic kidney; maximum number of binding sites; mitogen-activated protein kinase; parathyroid hormone receptor type 1; phenylephrine; phosphate-buffered saline with 0.1% Tween 20; polymerase chain reaction; prazosin; protein kinase A; protein kinase C; α(1) adrenoceptor; α(1)-AR; β(2) adrenoceptor; β(2)-AR